Apparatus and method for separating and concentrating fluids containing multiple components

ABSTRACT

An apparatus that allows for separating and collecting a fraction of a sample. The apparatus, when used with a centrifuge, allows for the creation of at least three fractions in the apparatus. It also provides for a new method of extracting the buffy coat phase from a whole blood sample. A buoy system that may include a first buoy portion and a second buoy member operably interconnected may be used to form at least three fractions from a sample during a substantially single centrifugation process. Therefore, the separation of various fractions may be substantially quick and efficient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/932,882, filed on Sep. 02, 2004, entitled “APPARATUS ANDMETHOD FOR SEPARATING AND CONCENTRATING FLUIDS CONTAINING MULTIPLECOMPONENTS”, which is a continuation-in-part of U.S. patent applicationSer. No. 10/445,381, filed on May 23, 2003, entitled “APPARATUS ANDMETHOD FOR SEPARATING AND CONCENTRATING FLUIDS CONTAINING MULTIPLECOMPONENTS” that claimed the benefit of U.S. Provisional Application No.60/383,013, filed on May 24, 2002. The disclosures of the aboveapplications are incorporated herein by reference.

FIELD

The present teachings relate to a multiple component fluid and aconcentrator/separator and, more particularly, relates to a containeroperable with a centrifuge to separate and/or concentrate variousbiological components.

BACKGROUND

Various fluids, such as whole blood or various other biological fluids,may be separated into their constituent parts, also referred to asfractions or phases. For example, whole blood samples may include aplurality of constituents that may be separated by density in a devicesuch as a centrifuge. The whole blood sample may be placed in a testtube, or other similar device, which is then spun in a centrifuge. Inthe centrifuge the whole blood is separated into different fractionsdepending upon the density of that fraction. The centrifugal forceseparates the blood or other sample into different fractions. Inaddition, various elements may be added to the test tube to create morethan two fractions. In particular, commonly used gels may be used todivide the whole blood into a plurality of different fractions which mayinclude fractions such as platelets, red blood cells, and plasma.Various other biological fluids may be separated as well. For example,nucleated cells may be separated and extracted from bone marrow oradipose tissue sample.

Many of these systems, however, do not provide a simple or efficientmethod to extract any more than one fraction and, especially, a fractionother than the top fraction. The top fraction of whole blood is plasma,or other blood constituents suspended in plasma. Thus, to extract otherfractions the plasma fraction must either be removed for furtherextracting procedures or spun again to obtain the constituents suspendedin this plasma. It is difficult to pierce the top fraction withoutcommingling the sample. Accordingly, obtaining the other fractions isdifficult with commonly known systems.

Other systems have attempted to alleviate this problem by providing afloat or other device that is disposed within the sample at theinterfaces of the different fractions during the centrifuge process.Nevertheless, these systems still do not allow a simple way to removethe different fractions without remixing the sample fractions. Inaddition, many of the systems do not allow an easy and reproduciblemethod to remove the desired sample fraction.

Therefore, it is desired to provide a device to allow for the easy andreproducible removal of a particular fraction which does not happen tobe the top fraction of a sample. It is desired to remove the requiredsample without mixing the different fractions during the extractionprocess. In addition, it is desired to provide a device which allows fora consistent extraction which includes known volumes or concentration ofthe fraction elements. Moreover, it is desired to separate andconcentrate a selected fraction with one centrifugation step.

SUMMARY

The present teachings provide an apparatus that separates andconcentrates a selected fraction or component of a fluid, such as abiological fluid. For example, undifferentiated cells, such asmesenchymal stem cells, platelet fraction, buffy coat, or white bloodcell fraction can be separated from bone reaming material, whole blood,bone marrow aspirate, and other materials. In various embodiments, theapparatus, when used with a centrifuge, is generally able to create atleast two fractions. The present teachings also provide for a new methodof creating at least three fractions extracting a third fraction from asample such as, for example, a buffy coat fraction.

In various embodiments, the apparatus includes a container to be placedin a centrifuge after being filled with a sample and the containerincludes a buoy or fraction separator having a selected density that maybe less than one fraction but greater than a second fraction, that isdisposed therein. In various embodiments, a second buoy may be placed inthe container with the first. The extraction system is connected to thebuoy system or to the collection chamber such that the fraction in thecontainer can be collected and drawn outside of the chamber. During thecentrifuge processing, the buoy is forced away from a bottom of thecontainer as the denser fraction collects at the bottom of thecontainer. The buoy is generally able to physically separate the denserfraction from another fraction of the sample. In various embodiments,the fractions can be withdrawn using an extraction system.

According to various embodiments, in addition to providing a first buoyand/or a second buoy, a buoy system may be provided. Generally, the buoysystem may separate the sample into at least three fractions. Thefractions may be separated and extracted from the container withoutsubstantially commingling the various fractions. Generally, a first buoyand a second buoy operate together to separate the sample into thevarious fractions and a syringe or tube may then be interconnected witha portion of the buoy system to extract the selected fractions. Forexample, a first buoy may be tuned to a density less than the density ofa red blood cell fraction of a whole blood sample, bone marrow aspiratesample, or combinations thereof, and a second buoy may be tuned to adensity less than the density of a buffy coat fraction.

According to various embodiments, a method of forming an enrichedscaffold for application relative to an anatomy is provided. The methodmay include obtaining a volume of a first whole material and obtaining avolume of a second whole material. A first fraction of the first wholematerial and a second fraction of the second whole material may beformed. At least one of the first fraction or the second fraction may beapplied to the scaffold and at least one fraction for applicationrelative to an anatomy is provided. The method may include obtaining avolume of heterogeneous whole material and separating the material intothe desired fraction(s). At least one of the fractions may be applied tothe scaffold.

According to various embodiments, a method of withdrawing a materialdirectly from a patient and collecting a selected fraction of thematerial in a container is provided. The method may include forming anaccess to port to the patient. A pressure differential in a collectioncontainer may be formed relative to the patient. A connection may bemade between the patient and the collection container via the port. Thecollection container may be filled with the material and then thematerial may be separated to form the selected fraction.

According to various embodiments, a method for concentrating boneaspirate can include obtaining a volume of bone marrow aspirate from amammal and loading the volume bone marrow aspirate into a separator, theseparator operable to separate the aspirate into three of morefractions. The method also includes centrifuging the separator to createa fraction that is a concentrated bone marrow aspirate and extractingand removing the fraction from the separator.

According to various embodiments, a method for concentrating bone marrowaspirate and blood includes collecting bone marrow aspirate and bloodfrom a patient then loading the bone marrow aspirate and blood into aseparator that can separate the aspirate and the blood into three ormore fractions. The method includes centrifuging the separatorcontaining the bone marrow aspirate and the blood creating a fractionthat has a concentrated bone marrow aspirate and a concentrated blood.In various embodiments, such a concentration may be referred to as abuffy coat. The method also include withdrawing the fraction comprisingthe concentrate or buffy coat.

According to various embodiments, a method for treating a defect in amammal using a concentrated bone marrow aspirate includes drawing bonemarrow aspirate from the mammal and loading the bone marrow aspirateinto a separator that can separate the bone marrow aspirate into threeor more fractions. The method includes centrifuging the separatorseparating the bone marrow aspirate into fractions and one fraction isconcentrated bone marrow aspirate. The method also can include theconcentrated bone marrow aspirate and applying the bone marrow aspirateto a site of a defect in the mammal.

According to various embodiments, a method for treating a defect in apatient includes drawing bone marrow aspirate and whole blood from thepatient then adding anticoagulants to the bone marrow aspirate and theblood. The method includes the loading of the bone marrow aspirate andthe blood into a separator that can separate the bone marrow aspirateand blood into three or more fractions. The method also includescentrifuging the separator then withdrawing a fraction comprising atleast one of the group consisting of hematopoietic stem cells, stromalstem cells, mesenchymal stem cells, endothelial progenitor cells, redblood cells, white blood cells, fibroblasts, reticulacytes, adiposecells, and endothelial cells, then applying the fraction to the site ofthe defect in the patient.

According to various embodiments, a method of treating a patient with acombination of concentrated bone marrow aspirate and buffy coat isprovided. The method includes obtaining blood and bone marrow aspiratefrom the patient, forming a buffy coat fraction of the whole blood andforming a concentrated bone marrow aspirate fraction, and applying atleast one of the buffy coat or concentrated bone marrow aspirates to thepatient.

Further areas of applicability of the present teachings will becomeapparent from the detailed description provided hereinafter. It shouldbe understood that the detailed description and specific examples, whileindicating various embodiment of the teachings, are intended forpurposes of illustration only and are not intended to limit the scope ofthe teachings.

BRIEF DESCRIPTION OF THE DRAWINGS

The present teachings will become more fully understood from thedetailed description and the accompanying drawings, wherein:

FIG. 1 is a plan view of a separator including a depth gage affixed to aplunger in a tube according to various embodiments of the presentteachings;

FIG. 2 is a cross-section view taken along line 2-2 of FIG. 1;

FIG. 3 is an exploded of the separator apparatus according to variousembodiments;

FIG. 4 is a kit including the separator according to an embodiment ofthe present teachings;

FIG. 5A is a plan view of the separator, according to variousembodiments, being filled;

FIG. 5B is a plan view of a blood sample in the separator after thecentrifuge process;

FIG. 5C is a plan view of the plunger plunged into the tube with thedepth gage to further separate the blood sample;

FIG. 5D is a plan view of the buffy coat and the plasma fractions beingextracted from the separator according to various embodiments;

FIG. 6A is a side plan view of a buoy system according to variousembodiments;

FIG. 6B is a cross-sectional view of the buoy system of FIG. 6 a;

FIG. 7A is a plan view of a separator, according to various embodiments,being filled;

FIG. 7B is a plan view of a separator, according to various embodiments,after a centrifugation process;

FIG. 7C is a plan view of a separator system, according to variousembodiments, being used to extract a selected fraction after thecentrifugation process;

FIG. 7D is a plan view of a second fraction being extracted from theseparator according to various embodiments;

FIG. 8 is a schematic view illustrating an assisted blood withdrawaldevice according to various embodiments; and

FIG. 9 is a block diagram illustrating a method for applying selectedfractions of a fluid.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

The following description of various embodiments is merely exemplary innature and is in no way intended to limit the teachings, itsapplication, or uses. Although the following description exemplaryrefers to a bone reaming material, whole blood and/or bone marrowaspirate separation, it will be understood that the present teachingsmay be used to separate and concentrate any appropriate material. Itwill be further understood that many multi-component materialscontaining particles may be separated. The components or fractions aregenerally intermingled in the whole sample but may be separated with acentrifuge device that causes increased local gravity or gravitationalforces.

With reference to FIGS. 1-3, according to various embodiments, aseparator 10, also referred to as a concentrator, is illustrated. Theseparator 10 generally includes a tube or container 12 that is adaptedto hold a fluid sample, such as an anti-coagulated whole blood sample,for further processing. It will be understood that the tube 12 may holdother solutions including constituents of more than one density, such asbone marrow or a mixture of whole blood and bone marrow. The tube 12includes a top or open end 12 a, which is closeable, and a bottom orclosed end 12 b. The bottom 12 b may also be selectively closeable.

Disposed within the tube 12 is a first piston or buoy 14 that is able tomove along a central axis A of the tube 12. The buoy 14 is generallynearer the bottom end 12 b of the tube 12 rather than the open end 12 a.The buoy 14 is able to move along a central axis A of tube 12. Alsodisposed within the tube 12 is a second piston or plunger 16. Theplunger 16 is also able to move within the tube 12 generally between aposition closer to the open end 12 a to a position closer to the closedend 12 b of the tube 12. A cap 18 substantially mates with the open end12 a of the tube 12 to close the tube 12 save for ports formed in thecap 18. Extending from the cap 18 is a plasma valve or port 20 thatcommunicates with an area, described further herein, within the tube 12defined between the plunger 16 and the cap 18. It will be understoodthat the plasma port 20 is merely exemplary in nature and simply allowsfor removal of a selected fraction of a sample such as, for example,plasma from whole blood.

The cap 18 also includes a depth gage port 19. Extending from theplunger 16 and through the depth gage port 19 is a first plunger port22. A depth guide or gage 24 includes a female connector 26 adapted toconnect with the first plunger port 22. The depth gage 24 also includesa depth gage housing or cannula 28. The depth gage housing 28 defines adepth gage bore 30. Incorporated in the depth gage housing 28 andextending distal from the end mating with the plunger 16 is a neck 32.The neck 32 includes external neck threads 34. The external neck threads34 are adapted to engage appropriate internal threads of a matingmember.

The mating member may include a compression nut 36 that mates with theexternal neck threads 34 to lock a depth gage rod 38 in a predeterminedposition. A split bushing 39 is also provided to substantially seal thedepth gage housing 28 when the depth gage rod 38 is locked in place. Thedepth gage rod 38 extends through the depth gage housing 28 andterminates at a rod handle 40. The rod handle 40 may be a form easilymanipulated by a human operator. The depth gage rod 38 extends coaxiallywith axis A of the tube 12. The depth gage rod 38 extends through theplunger 16 a predetermined distance and may be locked at that distancewith the compression nut 36.

Although the tube 12 is described herein as a cylinder, it will beunderstood that other shapes may be used, such as polygons. The internalportions, such as the cap 18, buoy 14, and plunger 16, would alsoinclude this alternate shape. Preferably the tube 12 is formed of athermal plastic material which is flexible under the forces required toseparate blood. The tube 12 may be made of a material that includes theproperties of both lipid and alcohol resistance. These properties helpsincrease the separation speed and decrease the amount of material whichmay cling to the tube wall 42. For example, Cyrolite MED2® produced byCyro Industries of Rockaway, N.J., may be used to produce the tube 12.

The tube 12 has a tube wall 42 with a thickness of between about 0.01millimeters and about 30.0 millimeters, although the tube wall 42 may beany appropriate thickness. The thickness of the tube wall 42 allows thetube wall 42 to flex during the centrifuge process yet be rigid enoughfor further processing of a blood sample disposed in the tube 12. Thetube 12 is closed at the bottom end 12 b with a tube bottom 44 formed ofthe same material as the tube wall 42 and is formed integrallytherewith. Generally, the tube bottom 44 has a thickness which issubstantially rigid under the forces required to separate the samplesuch that it does not flex.

The buoy 14 includes an upper or collection face 46 that defines aninverse cone or concave surface. Generally, the cone has an angle ofbetween about 0.5° to about 45°, and may be about 0.5° to about 90° froma vertical axis, wherein the apex of the cone is within the buoy 14. Thecollection face 46 forms a depression in the buoy 14 which collects andconcentrates material during the separation process. Additionally, thebuoy 14 has a bottom face 48 that defines an inverse cone, dome, orcovered surface. The buoy bottom face 48 includes an apex 50 thatengages the tube bottom 44 before a buoy edge 52 engages the tube bottom44. The buoy 14 includes a material that is a substantially rigid suchthat the buoy edges 52 never meet the tube bottom 44. Therefore, thereis a gap or free space 54 formed between the buoy edges 52 and the tubebottom 44 along the perimeter of the buoy 14.

The separator 10 is generally provided to separate a multi-componentfluid that generally includes various components or constituents ofvarying densities that are commingled or mixed together. The separator10 includes the buoy 14 that is of a selected density depending upon aselected constituent of the multi-constituent liquid. Although the buoy14 may be tuned or of any selected density, the following examplerelates to separation of whole blood to various components. Therefore,the buoy 14 will be discussed to include a selected density relative towhole blood separation. It will be understood, however, that the buoy 14may be of any appropriate density depending upon the multi-componentfluid being separated including, for example, the concentrating of bonemarrow aspirate.

The buoy 14 may be formed of any appropriate material that may have aselected density. For example, when the separator 10 is to separateblood, the buoy 14 generally has a density which is greater than that ofred blood cells in a whole blood sample, but less than the plasma ornon-red blood cell fraction of a whole blood sample. For blood, thedensity of the buoy 14 may be between about 1.00 g/cc to about 1.12 g/ccor between about 1.02 g/cc and about 1.09 g/cc.

To achieve the selected density, the buoy 14 may be formed as acomposite or multi-piece construction, including a plurality ofmaterials. Particularly, a first or outside portion 56 defines the upperor collection face 46 and the buoy edges 52 and is formed of the samematerial as the tube 12. The outside portion 56 defines a cup or voidinto which a plug or insert 58 is placed. The insert 58 has a mass suchthat the density of the entire buoy 14 is within the selected range, forexample, the range described above. Generally, a high densitypolyethylene may be used, but the material and size of the insert 58 maybe altered to produce the desired density of the buoy 14. Alternatively,the buoy 14 may be formed of a single suitable material that has adensity in the selected range. Nevertheless, the buoy 14 formedunitarily or of a single material would still include the other portionsdescribed in conjunction with the buoy 14.

The outside portion 56 of the buoy 14 also defines the outsidecircumference of the buoy 14. The outside circumference of the buoy 14is very close to the internal circumference of the tube 12. Due to theoperation of the buoy 14, however, described further herein, there is aslight gap between the outside of the buoy 14 and the inside of the tube12. Generally, this gap is between about 1 and about 10 thousandths ofan inch around the entire circumference of the buoy 14. Generally, it isdesired that the distance between the outside circumference of the buoy14 and the inside circumference of the tube 12 is great enough to allowa selected material or component to pass. For example, in whole bloodthe distance is selected so that red blood cells may pass through thegap without being lysed, damaged, or activated.

The plunger 16 includes a plunger front or collection face 60 and aplunger wall 62 that extends from the plunger front face 60. The plungerwall 62 extends relatively perpendicular to the plunger front face 60and substantially parallel to the tube wall 42. Extending from thecenter of the plunger 16 is a sample collection projection 64. Extendingfrom the top of the sample collection projection 64 is the first plungerport 22. The sample collection projection 64 includes a plunger samplecollection bore 68 defined therethrough. The plunger sample collectionbore 68 terminates at a sample collection aperture 70 that issubstantially in the center of the plunger front face 60. The plungerfront face 60 also defines an inverse cone where the sample collectionaperture 70 is the apex of the cone. The plunger front face 60 defines acone with an angle substantially similar or complimentary to thecollection face 46 of the buoy 14. In this way, the plunger front face60 may mate substantially completely with the collection face 46 forreasons described more fully herein.

The plunger 16 also includes a back face 72. Extending from the plungerfront face 60 to the back face 72 is a bore 74. A check valve 76 isoperably connected to the bore 74. The check valve 76 allows a liquid tomove from the plunger front face 60 to the back face 72 while notallowing the liquid to move from the back face 72 to the plunger frontface 60. Therefore, the check valve 76 is substantially a one-way valvewhich allows a material to move in only one direction. The check valve76 may also operate automatically allowing flow in only onepredetermined direction. Alternatively, the check valve 76 may beoperated manually and include a portion extending from the check valve76 requiring manipulation to stop or start a flow through the checkvalve 76.

The plunger 16 may be made out of any appropriate material which doesnot interfere with the separation of the fractions of the fluid, such aswhole blood. The plunger 16, however, is made of a material that isflexible or at least partially deformable. A flexible material allowsthe plunger 16 to have an external circumference defined by the plungerwalls 62 that is substantially equal to the internal circumference ofthe tube 12. Because of the deformability of the plunger 16, however,the plunger 16 is still able to move within the tube 12. The plunger 16is able to move through the tube 12 and also substantially wipe theinterior of the tube wall 42. This creates, generally, a moveable sealwithin the tube 12. Thus, substantially no material escapes the actionof the separator 10 when the plunger 16 is plunged into the tube 12.This also helps concentrate the portion of the sample desired to becollected, described more fully herein.

The cap 18 provides a structure to substantially close the tube 12. Thecap 18 particularly includes a plate 78 that has an externalcircumference substantially equal to the external circumference of thetube 12. Extending from the plate 78 and into the tube 12 is a flange80. The external circumference of the flange 80 is substantially equalto the internal circumference of the tube 12. In this way, the cap 18substantially closes the tube 12. It will be understood the cap 18 maybe in any form so long as the cap 18 substantially closes and/or sealsthe tube 12 when installed.

Formed through the center of the plate 78 is the depth gage port 19. Thedepth gage port 19 is also adapted to receive the sample collectionprojection 64. The first plunger port 22 extends above the plate 78through the depth gage port 19. The circumference of the depth gage port19 is substantially equal to the external circumference of the samplecollection projection 64 such that a liquid seal is formed. The plate 78defines a sample face 84 that includes an interior side of the cap 18.The area between the sample face 84 of the cap 18 and the back face 72of the plunger 16 define a plasma collection area 86. Although theplasma collection area 86 is exemplary called the plasma collectionarea, it will be understood that the plasma collection area 86 may alsocollect any appropriate fraction of the sample that is positioned withina separator 10. The plasma collection area 86 is merely an exemplaryname and an example of what material may be collected in the area of theseparator 10. As discussed herein, the separator 10 may used to separatewhole blood into various fractions, therefore, the plasma collectionarea 86 is used to collect plasma. The plasma collection area 86 alsoallows a space for the check valve 76 to be installed.

A second bore 88 is formed in the plate 78. Extending through the secondbore 88 is the plasma collection valve 20. In liquid communication withthe plasma collection valve 20 is a plasma collection tube 92. Theplasma collection tube 92 has a length such that the plasma collectiontube 92 is able to extend from the plasma collection valve 20 tosubstantially the tube bottom 44. The plasma collection tube 92,however, is flexible enough such that it may be folded or compressed tofit within the plasma collection area 86 when the plunger 16 issubstantially near the open end 12 a of the tube 12. The plasmacollection tube 92 may also be connected to a hose barb 93 that includesa plasma collection bore 93 a. The plasma collection bore 93 a issubstantially level with the plunger back face 72. Alternatively, theplasma collection bore 93 a may be positioned below the plunger backface 72 but in fluid communication with the plasma collection tube 92.

The outboard side of the plasma collection valve 20 may include externalthreads 94 to mate with internal threads of a plasma valve cap 96.Therefore, the plasma collection valve 20 may be selectively opened andclosed via the plasma valve cap 96. It will be understood, however, thatother appropriate means may be used to open and close the plasmacollection valve 20 such as a clip or a plug. It will be understood thatthe plasma collection valve 20, plasma collection tube 92, plasmacollection bore 23 a may be used to collect any appropriate material orfraction from the separator 10.

Also formed in the plate 78 is a vent bore 98. The vent bore 98 allowsair to flow into the plasma collection area 86 as the plunger 16 isbeing plunged into the tube 12. The vent bore 98 may include a filter100 such that liquid cannot escape from the tube 12. The filter 100allows air to enter or escape from the plasma collection area 86 whilemaintaining the liquid seal of the tube 12 produced by the cap 18.

Selectively attachable to the first plunger port 22 is the depth gage24. The female connector 26 interconnects the depth gage housing 28 tothe first plunger port 22. Internal threads in the female connector 26mate with an external thread 102 formed on the first plunger port 22. Itwill be understood, however, that other engagement mechanisms betweenthe depth gage 24 and the plunger 16 may be used. For example, a snapconnection rather than a threaded connection between the two may beused.

The depth gage housing 28 is formed to be substantially rigid. Suitablematerials, when sized properly, include polycarbonate and CYRO MED2®.The material preferably is both rigid and does not substantially reactwith the sample. It is rigid enough to provide a mechanism to plunge theplunger 16 into the tube 12. In addition, the external circumference ofthe depth gage housing 28 is substantially equal to the circumference ofthe depth gage port 19 in the plate 78. Therefore, as the plunger 16 isbeing plunged into the tube 12 with the depth gage 24, no liquidmaterial is allowed to escape around the depth gage housing 28 andthrough depth gage port 19.

Formed within the depth gage housing 28 is the bore 30 which receivesthe depth gage rod 38. The depth gage rod 38 extends through the plungersample collection bore 68 of the sample collection projection 64 andprotrudes through the sample collection aperture 70 a predeterminedlength. The depth gage rod 38 extends through the sample collectionaperture 70 a length such that when an end 104 of the depth gage rod 38meets the buoy 14, the volume defined by the collection face 46 and theplunger front face 60 is between about 5% and about 30% of the totalvolume of the sample that the tube 12 holds. The projection of the depthgage rod 38 allows for an easily reproducible collection amount andconcentration over several trials.

The compression nut 36 locks the depth gage rod 38 in the predeterminedposition. Nevertheless, once the plunger 16 has been plunged to thedesired depth in the tube 12, the compression nut 36 may be loosened sothat the depth gage rod 38 may be removed from the plunger 16 and thedepth gage housing 28 without moving the plunger 16. A syringe or otherappropriate device may then be affixed to the external neck threads 34of the depth gage 24 to extract the fraction or phase that is betweenthe plunger front face 60 and the collection face 46. As describedfurther herein, the fraction or phase that is left between the plungerfront face 60 and the collection face 46 may be the buffy coat of awhole blood sample. Nevertheless, it will be understood that thefraction between the plunger front face 60 and the collection face 46may be any appropriate fraction of the sample that is disposed in theseparator 10.

The separator 10 may be provided alone or in a kit 200, as illustratedin FIG. 4. The kit 200 may be placed in a tray 202 which is covered toprovide a clean or sterile environment for the contents of the kit 200.The kit 200 may include at least a first separator 10 and a secondseparator 10′. A first depth gage 24 and a second depth gage 24′ arealso provided, one for each separator 10, 10′. The kit 200 alsogenerally includes a first syringe 204, including a needle, to draw abiological sample, such as blood from a patient. The first syringe 204may also be used to place the sample in the first separator 10. Aftercentrifuging the sample a second device or syringe 210 may be used toextract a first fraction of the sample, while a third device or syringe212 may be used to extract a second fraction of the sample. Also, atourniquet 214 and other medical supplies, such as gauze 216 and tape218, may be provided to assist the practitioner. It will be understoodthe elements of the kit 200 are merely exemplary and other appropriateitems or elements may be included.

With reference to FIGS. 5A-5D, a method using the separator 10 isillustrated according to various embodiments. The following examplerelates specifically to the taking and separating of a sample of wholeblood from a patient. Nevertheless, it will be understood that anotherappropriate biological material may be separated and concentrated usingthe separator 10. For example, bone marrow may be separated andconcentrated using the separator 10. The various fractions of the bonemarrow are similar to the fractions of whole blood. Generally, the bonemarrow includes a fraction that includes substantially dense materialand a second phase that is less dense and has other components suspendedtherein such as, for example, nucleated cells. The bone marrow samplemay be positioned in the separator 10, similarly to the whole blood asdescribed herein, and separated in a substantially similar manner as thewhole blood. The separator 10 can then be used to remove nucleated cellsfrom the bone marrow sample (which may be referred to as buffy coat),whereas the separator 10, as described herein, is used to remove thebuffy coat from the whole blood which includes platelets and otherappropriate materials (which may be referred to as platelet rich plasma(PRP)).

A mixture of whole blood and bone marrow may be positioned in theseparator 10 for separation and concentration. Similar methods and stepswill be used to separate the mixture of whole blood and bone marrow witha main difference being the material that is separated. It will also beunderstood that various centrifuge times or forces may be altereddepending upon the exact material that is being separated with theseparator 10. It will also be understood that the separation of wholeblood, bone marrow, or a mixture of whole blood and bone marrow aremerely exemplary of the materials that may be separated using theseparator 10.

According to various embodiments, and with reference to FIGS. 5A-5D andto a whole blood sample, a sample of whole blood taken from a patient isplaced in the tube 12 with an anticoagulant using the first syringe 204or other appropriate delivery method. In particular, the first syringe204 may be connected to the first plunger port 22. After which the bloodsample is provided to the tube 12 via the sample collection bore 68 andsample collection aperture 70. A cap 220 is then placed over the firstplunger port 22 to substantially seal the tube 12.

After the whole blood sample is delivered to the tube 12, the separator10 is placed in a centrifuge. The second separator 10′, substantiallyidentical to the first, is placed opposite the first separator 10including the sample in a centrifuge. The second separator 10′ may alsoinclude a second sample or may include a blank, such as water, so thatthe centrifuge is balanced. The second separator 10′ balances thecentrifuge by both weight and dynamics.

The separator 10 is then spun in the centrifuge in a range between about1,000 and about 8,000 RPM. This produces a force between about 65 andabout 4500 times greater than the force of normal gravity, as generallycalculated in the art, on the separator 10 and the blood sample placedin the separator 10. At this force, the more dense material in a wholeblood sample is forced toward the bottom end 12 b of the tube 12. Thedense material, such as red blood cells or a red blood cell fraction222, collects on the tube bottom 44. Because the buoy 14 has a densitythat is less than the red blood cell fraction 222, it is forced in adirection toward the top end 12 a of the tube 12 in the centrifuge.Nevertheless, because the buoy 14 is denser than a plasma fraction 224,the buoy 14 does not reach the top end 12 a of the tube 12.

The forces also affect the tube wall 42. The forces compress the tube 12linearly along axis A thereby bowing or flexing the tube wall 42. As thetube wall 42 compresses it increases the diameter of the tube 12 makingit easier for the buoy 14 to move in the direction of the top 12 a ofthe tube 12. In addition, the bottom face 48, defining an inverse cone,helps the initial movement of the buoy 14. Because the buoy 14 is notsubstantially flat along its bottom, it does not form a vacuuminteraction with the tube bottom 44. Therefore, the initial movement ofthe buoy 14 away from the tube bottom 44 is quicker than if the bottomof the buoy 14 was flat.

During the centrifuge process, the red bloods cells of the red bloodcell fraction 222 force the buoy 14 in the direction of the top end 12 aof the tube 12 because the buoy 14 is less dense than the red blood cellfraction 222. Although the whole blood sample, including the red bloodcells, is loaded above the buoy 14, the red blood cells are able to movebetween the buoy 14 and the tube wall 42 because the circumference ofthe buoy 14 is less than the internal circumference of the tube 12.During the centrifuge process, the buoy 14 stops at an interface of aplasma fraction 224 and the red blood cell fraction 222 because of theselected or tuned density of the buoy 14.

With particular reference to FIG. 5B, the centrifuge process has beencompleted and the buoy 14 has moved to the interface of the red bloodcell fraction 222 and plasma fraction 224. After the centrifuge hasslowed or stopped, and before or after the tube 12 has been removed fromthe centrifuge, the tube wall 42 decompresses which helps support thebuoy 14 at the interface position. It is also understood that applyingan external pressure to the tube 12 via fingers or another apparatus mayhelp stabilize the buoy 14 during the plunging procedure describedherein.

On or near collection face 46 is a middle fraction 226, including asmall, yet concentrated, amount of red blood cells, white blood cells,platelets, and a substantial portion of a buffy coat of the bloodsample. Although the plasma is also present near the collection face 46at this point, the solid portions of the buffy coat are more compressedagainst the collection face 46. The position of the buoy 14 also helpsin this matter. Because the buoy 14 is a single body it defines theinterface of the plasma fraction 224 and the red blood cell fraction222. Also the density of the buoy 14 assures that it has not passed intothe plasma fraction 224. Therefore, the fractions remain separated afterthe centrifuge process. In addition because the buoy 14 is tuned to thedensity of the red blood cell fraction 222, it is not affected byvariations in the density of the plasma fraction 224 and the buoy's 14position is always at the interface of the red blood cell fraction 222and the plasma fraction 224.

With particular reference to FIG. 5C, the depth gage 24 is affixed tothe first plunger port 22 of the sample collection projection 64. Afterconnecting the depth gage 24 to the first plunger port 22, the plunger16 is plunged into the tube 12 by pushing on the depth gage 24. As thisis performed the plasma fraction 224, formed and separated above thebuoy 14, is able to flow through the check valve 76 into the plasmacollection area 86. This displacement of the plasma fraction 224 allowsthe plunger 16 to be plunged into the tube 12 containing the bloodsample.

The plunger 16 is plunged into the tube 12 until the point where the end104 of the depth gage rod 38 reaches the buoy 14. The volume left in thecollection face 46 is the middle fraction 226 and is determined by thedepth gage 24. It may be adjusted by selectively determining the amountthat the depth gage rod 38 extends below the plunger front face 60. Byadjusting the depth gage 24, the concentration of the middle fraction226 can be adjusted depending upon the desires of the operator.

The plasma fraction 224 is held in the plasma collection area 86 forlater withdrawal. Therefore, the use of the plunger 16 and the buoy 14creates three distinct fractions that may be removed from the tube 12after only one spin procedure. The fractions include the red blood cellfraction 222, held between the buoy 14 and the tube bottom 44. Themiddle or buffy coat fraction 226 is held between the plunger 16 and thebuoy 14. Finally, the plasma fraction 224 is collected in the plasmacollection area 86. In various embodiments, the middle fraction 226 maybe a platelet rich plasma (PRP) fraction and the plasma fraction 224 maybe a platelet poor plasma (PPP) fraction.

The middle fraction 226 may be extracted from the tube 12 first, withoutcommingling the other fractions, through the sample collection bore 68.With particular reference to FIG. 5D, the depth gage rod 38 may beremoved from the depth gage housing 28. This creates a sample collectioncannula which includes the depth gage bore 30, the sample collectionbore 68, and the sample collection aperture 70. After the depth gage rod38 has been removed, the second syringe 210 may be affixed to the depthgage housing 28 via the external neck threads 34. The second syringe 210may be substantially similar to the first syringe 204.

Before attempting to withdraw the middle fraction 226 the separator 10may be agitated to re-suspend of the platelets and concentrated redblood cells in a portion of the plasma remaining in the collection face46. This allows for easier and more complete removal of the middlefraction 226 because it is suspended rather than compressed against thecollection face 46. A vacuum is then created in the second syringe 210by pulling back the plunger 16 to draw the middle fraction 226 into thesecond syringe 210.

As the middle fraction 226 is drawn into the second syringe 210 theplunger 16 moves toward the buoy 14. This action is allowed because ofthe vent bore 98 formed in the cap 18. Atmospheric air is transferred tothe plasma collection area 86 through the vent bore 98 to allow themiddle fraction 226 to be removed. This also allows the movement of theplunger 16 toward the buoy 14. This action also allows the plunger 16 to“wipe” the collection face 46. As the plunger front face 60 mates withthe collection area 46 the middle fraction 226 is pushed into the samplecollection aperture 70. This ensures that substantially the entiremiddle fraction 226 collected in the collection area 46 is removed intothe second syringe 210. It can also increase the repeatability of thecollection volumes. In addition, because the second syringe 210 does notprotrude out the sample collection aperture 70, it does not interferewith the collection of the middle fraction 226. Once the plunger frontface 60 has mated with the collection face 46 there is substantially novolume between the plunger 16 and the buoy 14.

Once the middle fraction 226 is extracted the second syringe 210 isremoved from the first plunger port 22. Also the extraction of themiddle fraction 226 leaves the plasma fraction 224 and the red bloodcell fractions 222 separated in the tube 12. At this point, a thirdsyringe 212 may be affixed to the plasma collection valve 20. The thirdsyringe 212 is connected to the external threads 94 of the plasmacollection valve 20 to ensure a liquid tight connection. It will beunderstood, however, that another connection mechanism such as a snap orcompression engagement may be used to connect the third syringe 212 tothe plasma collection valve 20.

A vacuum is then created in the third syringe 212 to draw the plasmafraction 224 from the plasma collection area 86 through the plasmacollection tube 92. As discussed above, the plasma collection tube 92 isconnected to the hose barb 93. Therefore, the plasma flows through theplasma collection bore 93 a through the hose barb 93, and then throughthe plasma collection tube 92. It will be understood that the plasmacollection tube 92 may alternatively simply rest on the plunger backface 72 to collect the plasma fraction 224. In this way, the plasmafraction 224 may be removed from the blood separator 10 withoutcommingling it with the red blood cell fraction 222. After the plasmafraction 224 is removed, the separator 10 may be dismantled to removethe red blood cell fraction 222. Alternatively, the separator 10 may bediscarded in an appropriate manner while retaining the red blood cellfraction 222.

The separator 10 allows for the collection of three of a whole bloodsample's fractions with only one centrifugation spin. The interaction ofthe buoy 14 and the plunger 16 allows a collection of at least 40% ofthe available buffy coat in the whole blood sample after a centrifugeprocessing time of about 5 minutes to about 15 minutes. Thecomplimentary geometry of the plunger front face 60 and the collectionface 46 help increase the collection efficiency. Although only the conegeometry is discussed herein, it will be understood that various othergeometries may be used with similar results.

The plunger front face 60 being flexible also helps ensure a completemating with the collection face 46. This, in turn, helps ensure thatsubstantially the entire volume between the two is evacuated. Theprocess first begins with the suction withdrawal of the middle fraction226 via the second syringe 210, but is completed with a fluid forceaction of the middle fraction 226 as the plunger front face 60 mateswith the collection face 46. As the plunger front face 60 mates with thecollection face 46, the fluid force assists in removal of the selectedfraction.

The plunger 16 also substantially wipes the tube wall 42. Because theplunger 16 is formed of a flexible material it forms a seal with thetube wall 42 which is movable. Therefore, substantially no liquid isable to move between the plunger wall 62 and the tube wall 42. Materialis substantially only able to go past the plunger front face 60 via thecheck valve 76.

The complimentary geometry also helps decrease the collection time ofthe middle fraction 226. Therefore, entire time to prepare and removethe middle fraction 226 is generally about 5 to about 40 minutes. Thisefficiency is also assisted by the fact that the separator 10 allows forthe removal of the middle fraction 226 without first removing the plasmafraction 224, which includes the buffy coat, and re-spinning the plasmafraction 224. Rather, one spin in the separator 10 with the whole bloodsample allows for the separation of the buffy coat for easy extractionthrough the plunger 16.

As discussed above, the separator 10 may be used to separate anyappropriate multi-component material. For example, a bone marrow samplemay be placed in the separator 10 to be centrifuged and separated usingthe separator 10. The bone marrow sample may include several fractionsor components that are similar to whole blood fractions or may differtherefrom. Therefore, the buoy 14 may be altered to include a selecteddensity that is dependent upon a density of a selected fraction of thebone marrow. The bone marrow may include a selected fraction that has adifferent density than another fraction and the buoy 14 may be designedto move to an interface between the two fractions to allow for aphysical separation thereof. Similar to the whole blood fraction, theplunger 16 may then be moved to near a collection face 46 of the buoy14. The fraction that is then defined by the collection face 46 and theplunger 16 may be withdrawn, as described for the removal of the buffycoat from the whole blood sample. For example, the middle fraction 226in the bone marrow sample may include a fraction of undifferentiated orstem cells. In various embodiments, the middle fraction 226 in a bonemarrow sample may include hematopoietic, stem cells, stromal stem cells,mesenchymal stem cells, endothelial progenitor cells, red blood cells,white blood cells, fibroblasts, reticulacytes, adipose cells, orendothelial cells. In various embodiments, the middle fraction 226 isconcentrated bone marrow aspirate.

It will also be understood that mixtures of various fluids may beseparated in the separator 10. For example, a mixture of whole blood andbone marrow may be positioned in the separator 10 at a single time. Thebuoy 14 may be tuned to move to an interface that will allow for easyremoval of both the buffy coat, from the whole blood sample, and theundifferentiated cells, from the bone marrow sample. Nevertheless, itwill be understood that the separator 10 may be used within anyappropriate biological material or other material having multiplefractions or components therein. Simply, the buoy 14 may be tuned to theappropriate density and the plunger 16 may be used to cooperate with thebuoy 14 to remove a selected fraction.

According to various embodiments and with reference to FIGS. 6A and 6B,a buoy system 300 is illustrated. The buoy system 300 generally includesa first buoy or fraction separator member 302 and a second buoy memberor fraction separator 304. The first buoy 302 and the second buoy 304may be operably interconnected with a buoy system cylinder or member306. The buoy system 300 may be placed in a tube, such as the tube 12.The tube 12 may be formed of any appropriate material, such as theCryolite Med®2 as discussed above. Nevertheless, the buoy system 300 maybe designed to fit in the tube 12 or may be formed to fit in anyappropriate member that may be disposed within a selected centrifugingdevice. It will be understood that the following discussion relating tobuoy system 300 to be substantially matched to the size of the tube 12is merely exemplary. As the buoy 14 may be sized to fit in anyappropriate tube, the buoy system 300 may also be sized to fit in anyappropriate tube. It will be further understood that the tube 12 may beany appropriate shape. The tube 12 need not only be cylindrical but mayalso be or include conical portions, polygonal portions, or any otherappropriate shapes.

The first buoy 302 of the buoy system 300 may be generally similar ingeometry to the buoy 14. It will be understood that the first buoymember 302 may be formed in the appropriate manner including shape orsize to achieve selected results. Nevertheless, the first buoy member302 generally includes an exterior diameter that may be slightly smallerthan the interior diameter of the tube 12. Therefore, the first buoymember 302 may be able to move within the tube 12 during the centrifugalprocess. Also, as discussed above, the tube 12 may flex slightly duringthe centrifuging process, thus allowing the first buoy member 302 toinclude an exterior diameter substantially equivalent to the interiordiameter of the tube 12. As discussed further herein, during thecentrifugation process, a portion of the fraction of a sample may passbetween the exterior wall of the first buoy member 302 and the tube 12.

The first buoy member 302 may generally include a density that issubstantially equivalent to a first or selected fraction of the sample.If the sample to be separated includes whole blood and is desired toseparate the red blood cells from the other portions of the sample, thefirst buoy member 302 may have a selected density that may be about 1.00grams per cc (g/cc) to about 1.10 g/cc. It will be understood that thedensity of the first buoy member 302 may be any appropriate density,depending upon the fraction to be separated, and this range of densitiesis merely exemplary for separating red blood cells from a whole bloodsample.

In addition, the first buoy member 302 includes a collection face orarea 308 at a proximal or upper portion of the first buoy member 302.The collection face 308 generally defines a concave area of the firstbuoy member 302 and may have a selected angle of concavity. The buoyassembly 300 defines a central axis D. The collection face 308 defines asurface E that is formed at an angle γ to the central axis D of the buoysystem 300. The angle γ may be any appropriate angle and may be about0.5° to about 90°. The angle γ may, however, be between about 45° and89.5°. Nevertheless, it will be understood that the angle γ may be anyappropriate angle to assist in collection of a selected fraction orportion of the sample by the first buoy member 302.

A bottom or lower surface 310 of the first buoy member 302 may define abottom face. The bottom face 310 may also be formed at an angle Drelative to the central axis D. The bottom surface 310 defines a surfaceor plane F that may be formed at an angle Δ relative to the central axisD of the buoy system 300. The angle Δ may be any appropriate angle andmay be about 90° to about 160°. For example, the angle Δ may be about15°. Similarly to the buoy bottom face 48, the bottom surface 310defines an apex 312 that may first engage the bottom 12d of the tube 12,such that most or the majority of the bottom surface 310 does not engagethe tube 12. As illustrated further herein, the apex 312 allows for afree space or gap to be formed between the bottom face 310 of the firstbuoy member 302 and the bottom 12 b of the tube 12.

The second buoy member 304 may include an outer diameter substantiallyequivalent to the outer diameter of the first buoy member 302.Therefore, the second buoy 304 may move with the first buoy 302,particularly if the second buoy 304 is interconnected with the firstbuoy 302 with the buoy central cylinder 306. Nevertheless, the secondbuoy member 304 may be allowed to move substantially freely within thetube 12 during the centrifuging process.

The second buoy member 304 also includes an upper or superior surface314 that defines a plane G that is formed at an angle relative to thecentral axis D of the buoy system 300. The angle ε of the plane Grelative to the central axis D of the buoy system 300 may be anyappropriate angle. For example, the angle ε may be about 90° to about150°. Generally, the angle ε may assist in allowing a selected fractionor a portion of the sample to pass over the top surface 314 and past thesecond buoy member 304 during the centrifuging process.

The second buoy member 304 also define a bottom or inferior surface 316that also defines a plane H that may be formed at an angle K relative tothe central axis D of the buoy system 300. The angle K may be anyappropriate angle, such as about 90° to about 150°. Nevertheless, theangle K may be substantially complimentary to the angle γ of thecollection face 308 of the first buoy member 302. For example, if theangle γ is about 80°, the angle K may be about 100°, such thatsubstantially 180° or a straight line is formed when the first buoymember 302 engages the second buoy member 304. This may be for anyappropriate reason, such as extraction of a fraction that may bedisposed near the collection face 308 of the first buoy member 302.Nevertheless, the angle K may be any appropriate angle as the angle γ.

The second buoy member 304 may be formed to include any appropriatedensity. For example, the second buoy member 304 may include a densitythat is less than the plasma fraction of a whole blood sample. It willbe understood that the second buoy member 304 may include anyappropriate density and a density that is less than the plasma fractionof a whole blood sample is merely exemplary. Nevertheless, if a wholeblood sample is desired to be separated and the plasma sample is to besubstantially separated from another fraction, the second buoy member304 may include a density that is less than the plasma fraction of thewhole blood sample. Therefore, the density of the second buoy member 304may be about 0.01 g/cc to about 1.03 g/cc. As described herein, if thesecond buoy member 304 includes a density less than the plasma fractionof a whole blood sample and the first buoy member 302 includes a densitygreater than that of the red blood cells, the buoy system 300 may besubstantially positioned near an interface between the red blood cellfraction and the plasma fraction of a whole blood sample. Therefore, asdiscussed above, and further described herein, the platelet or buffycoat fraction of the whole blood sample may be substantially collectednear or in the collection face 308 of the buoy system 300.

The buoy post 306 may operably interconnect the first buoy member 302and the second buoy member 304. The buoy post 306 may be any appropriateconnection member. The buoy post need not be a single cylindricalportion. For example the buoy post 306 may include one or more membersinterconnecting the first buoy member 302 and the second buoy member304, such as around a perimeter thereof. In addition, the buoy post 306may include any appropriate shape or geometry.

The buoy system post 306 may be rigidly affixed to the first buoy member302 and the second buoy member 304, such that the first buoy member 302may not move relative to the second buoy member 304 and vice versa.Alternatively, the buoy post 306 may be slide ably connected to eitheror both the first buoy member 302 and the second buoy member 304.According to various embodiments, the buoy post 306 is generally fixedlyconnected to the first buoy member 302 and slide ably interconnected tothe second buoy member 304. The buoy post 306 may include a catchportion or lip 320 that is able to engage a portion of the second buoymember 304, such that a range of travel of the second buoy member 304,relative to the first buoy member 302 is limited. Nevertheless, therange of travel of the second buoy member 304 toward the first buoymember 302 may be substantially unlimited until the second buoy member304 engages the first buoy member 302.

In various embodiments, the buoy post 306 may also define a centralcannula or bore 322. The post bore 322 may include a connection portion324 substantially defined near an upper or a proximal end of the buoypost 306. This may allow for interconnection of various components withthe buoy post 306, such that various components may be moved through thebore 322 from an exterior location. The buoy post 306 may also define aport or cannula 326 that connects the post cannula 322 with thecollection face 308. Therefore, a substance may travel through the postcannula 322 and through the port 326. Various substances may then beprovided to or removed from the collection face 308 of the first buoymember 302.

In various embodiments, the buoy system 300 may be used to separate aselected multi component sample, such as a whole blood sample. Withcontinuing reference to FIGS. 6A and 6B, and reference to FIGS. 7A-7D, amethod of using the buoy system 300, according to various embodiments,is illustrated and described. With reference to FIGS. 7A-7D, likereference numerals are used to indicate like portions of the tube 12 andthe associated mechanisms described in FIGS. 1-3. Therefore, it will beunderstood that the buoy system 300 may be used with the tube 12 or anyother appropriate tube or container system or apparatus. Nevertheless,for simplicity, the description of a method of use of the buoy system300 will be described in conjunction with the tube 12.

The tube 12 may include the cap 18 that further defines a plasma valveor port 20. Extending through the cap 18 and interconnecting with afirst flexible tube or member 92, the plasma port 20 may be used toextract a selected fraction of the sample that is positioned above thesecond buoy member 304. The tube 12 may also define a second port 21,which may also be referred to as a platelet rich plasma (PRP) port. Asdiscussed herein, a second flexible member, such as a flexible tube 21a, may interconnect the PRP port 21 and a connection portion 324 of abuoy cylinder 306. As illustrated above, the first tube 92 may also beinterconnected with a selected portion of the system, such as the topsurface 314 of the second buoy member 304. As illustrated above, a valvemay be positioned and is operably interconnect the tube 92 with theupper surface 314 of the second buoy member 304. Nevertheless, such avalve is not necessary and it may be provided merely for convenience.

Other portions of the blood separator system 20, particularly thoseportions of the tube 12 and the cap 18 that have various valvesconnected therewith may be included in the tube 12 and used with thebuoy system 300. Nevertheless, once the buoy system 300 isinterconnected, it may be positioned in the interior of the tube 12 andthe syringe 204 used to place a sample into the tube 12. The sample maybe expressed from the syringe 204 into the interior of the tube 12 andthe sample may be any appropriate sample, such as a whole blood sample.Nevertheless, it will be understood, such as discussed above, variousother samples may be used, such as bone marrow samples, a mixture ofbone marrow and whole blood or non-biological fluids or materials. Itwill be understood that two buoys 302 and 304 may generally be near oneanother when the sample is positioned in the tube 12, but areillustrated apart for clarity of the present discussion.

Also, the sample may be placed in the tube 12 according to variousembodiments. As described above, an anticoagulant or other componentsmay be mixed with the whole blood sample, if a whole blood sample isused, before the whole blood sample is positioned within the tube 12. Asis apparent to one skilled in the art, an anticoagulant or othercomponents may be mixed with a bone marrow aspirate sample or a sampleof bone marrow aspirate and whole blood, if such samples are used beforesuch sample is positioned within the tube 12. The syringe 204 isconnected with the plunger port 22 extending from the cap 18, although aplunger may not be used in various embodiments.

After the sample is positioned within the tube 12, as described above, acap may be positioned over the port 22, such that the sample is notallowed to escape from the tube 12. After the sample is placed in thetube 12 and the cap placed on the port 22, the tube 12 including thesample and the buoy system 300 may be centrifuged.

With reference to FIG. 7B, after a centrifugation of the tube 12,including the buoy system 300, substantially three fractions of thesample may be formed. A first fraction 330 may be positioned between thebottom face 310 and the bottom of the tube 44. A middle fraction 332 maybe positioned between the collection face 308 and the bottom surface 316of the second buoy 304. In addition, a third fraction 334 may bepositioned between the upper surface 314 and the cap 18 of the tube 12.Generally, the first fraction 330, the middle fraction 332, and thethird fraction 334 are substantially physically separated with the buoysystem 300. During the centrifugation process, the tube 12 may flexslightly to allow for ease of movement of the buoy system 300 throughthe tube 12 and the sample. Nevertheless, the buoy system 300, duringthe centrifugation process, substantially creates the three fractions330, 332, and 334 without the operation of an operator. Therefore, theformation of at least three fractions may be substantially simultaneousand automatic using the buoy system 300.

The buoy system 300 substantially separates the fractions 330, 332, and334, such that they may be easily removed from the tube 12. For example,with reference to FIG. 7C, a syringe or other instrument 340 may be usedto extract the middle fraction 332 by interconnecting a cannula or boredtube 342 with the connection portion 324 of the buoy cylinder 306. Bydrawing the plunger 344 into the extraction syringe 340, a vacuum orupward force is produced within the extraction syringe 340. This forcedraws the middle fraction 332 through the ports 326 of the buoy post 306and through the buoy cannula 322. Therefore, the middle fraction 332 maybe extracted from the tube 12 without substantially commingling themiddle fraction 332 with either the first fraction 330 or the thirdfraction 334. The middle fraction 332 is drawn in the direction of arrowM through the cannula 322 and into the extraction syringe 340.

It will be understood that the second tube 21 a may also be used. Theextraction syringe 340 may be interconnected with the PRP port 21 thatis interconnected with the connection portion 324 of the buoy cylinder306. As discussed herein, the buoy cylinder allows access to the middlefraction 332 (platelet rich portion) between the buoy portions. Thus, itwill be understood, that access may be obtained and the middle fraction332 (platelet rich portion or buffy coat of the sample), between the twobuoys, may be extracted in a plurality of ways. The illustrations andmethod described herein is merely exemplary. For example, if bone marrowaspirate is used as the sample, the PRP port 21 would allow forextraction of undifferentiated nucleated cells. In various embodiments,the PRP port 21 allows for extraction of the buffy coat.

Alternatively, if the post 306 is not provided other portions may beprovided to gain access to the middle fraction 332. For example, if aplurality of members are provided around the perimeter of the first buoy302 and the second buoy 304 a valve portion, such as a puncture-ablevalve, may be provided in the second buoy 304 to be punctured with anobject. In this way an extraction needle may puncture the valve to gainaccess to the middle fraction 332. Regardless, it will be understoodthat the buoy system 300 may be able to form a plurality of fractions,such as the three fractions 330, 332, and 334 and at least the middlefraction 332 may be extracted without substantially commingling thevarious fractions.

During the extraction of the middle fraction 332 through the cannula322, the second buoy member 304 may move in the direction of arrow Mtoward the first buoy member 302. As described above, the collectionface 308 of the first buoy member may include an angle γ that issubstantially complementary to the bottom face 316 of the second buoymember 304. Therefore, if the second buoy member 304 is allowed to movealong the buoy cylinder 306, the bottom face 316 of the second buoymember 304 may be able to substantially mate with the collection face308 of the first buoy member 302. Alternatively, if the second buoymember 304 is not allowed to move, the second buoy member may beprovided with a vent port or valve, such that the extraction of themiddle fraction 332 from the collection face 308 may not be hindered bythe buildup of undesirable forces. Nevertheless, if the second buoymember 304 may move, the interaction of the bottom face 316 of thesecond buoy member 304 may assist in substantially removing the entiremiddle fraction 332 from the tube 12. As described above, the bottomface 60 of the plunger 16 may also serve a similar purpose when engagingthe collection face 46 of the buoy 14.

With reference to FIG. 7D, once the middle fraction 332 has beenextracted from the tube 12, the second buoy member 304 may substantiallymate with a portion of the first buoy member 302. As discussed above,the second buoy member 304 may substantially only mate with the firstbuoy member 302 if the second buoy member 304 is able to substantiallymove relative to the first buoy member 302. Therefore, it will beunderstood that the second buoy member 304 need not necessarily matewith the first buoy member 302 and is merely exemplary of an operationof various embodiments. Nevertheless, once the middle fraction 332 hasbeen extracted from the tube 12, the port 20 may be used in conjunctionwith a selected instrument, such as a plasma extraction syringe 212 toremove the plasma or the third fraction 334 from the tube 12 using theextraction tube 92 interconnected with the port 20.

As described above, the tube 92 allows for extraction of the thirdfraction 334 from the tube 12 without commingling the third fraction 334with the remaining first fraction 330 in the tube 12. Therefore, similarto the separator and extraction system 10, three fractions may besubstantially formed within the tube 12 with the buoy system 300 and maybe extracted without substantially commingling the various fractions.Once the third fraction 334 is extracted from the tube 12, the buoysystem 300 may be removed from the tube 12, such that the first fraction330 may be removed from the tube 12. Alternatively, the first fraction330 may be discarded with the tube 12 and the buoy system 300 as adisposable system. Alternatively, the system may be substantiallyreusable, such that it can be sterilized and may be sterilized forvarious uses.

The description of the method of use of the buoy system 300 is exemplaryof a method of using a system according to various other embodiments. Itwill be understood, however, that various specifics may be used fromvarious embodiments to allow for the extraction of selected fractions.For example, the centrifugation process may be substantially a singlestep centrifugation process. The buoy system 300, according to variousembodiments, may allow for the formation of three fractions during asingle centrifugation process. This centrifugation process may occur atany appropriate speed, such as about 1000 RPM to about 8000 RPM. Thisspeed may produce a selected gravity that may be approximately 4500times greater than the normal force of gravity. Nevertheless, thesespecifics are not necessary to the operation of the buoy system 300according to various embodiments. The buoy system 300, according tovarious embodiments, may be used to extract a plurality of fractions ofa sample after only a single centrifuging process and withoutsubstantially commingling the various fractions of the sample.

With reference to FIG. 8, the blood collection and separation systemthat includes the tube 12, according to various embodiments, may befilled with a multi-component fluid or solution, such as blood from apatient, is illustrated. The tube 12 may include any appropriateseparation system, such as the separation system 300. Nevertheless, inaddition to filling the tube 12 with a fluid from the syringe 204 anyappropriate method may be used to fill the tube 12. For example, when asolution, including a plurality of components, is placed into the tube12 it may be collected directly from a source.

For example, a patient 350 may be provided. The patient 350 may beprovided for a selected procedure, such as generally an operativeprocedure or other procedure that requires an intravenous connection352, such as a butterfly needle, to be provided in the patient 350. Theintravenous connection 352 generally provides a tube 354 extendingtherefrom. The tube 354 may be used to withdraw fluids from the patient350 or provide materials to the patient 350, such as medicines or otherselected components. Nevertheless, the intravenous connection 352 isgenerally provided for various procedures and may be used to fill thetube 12.

The tube 354 may interconnect with the plunger port 22 or anyappropriate portion of the tube 12. The port 22 may be used to connectwith the tube 354 in a similar manner as it would connect with thesyringe 204, if the syringe 204 was provided. Nevertheless, it will beunderstood that the tube 354 may be provided directly to the tube 12from the patient 350. This may reduce the number of steps required tofill the tube 12 and reduce possible cross-contamination from thepatient 350 with the various components. Moreover, making a connectiondirectly with the patient 350 may make the withdrawal and collection ofblood from the patient 350 more efficient.

Once the tube 354 is interconnected with the tube 12 the pressuredifferential between the patient 350, such as the intravenous pressureof the blood, may be used to fill the tube 12 to a selected volume. Inaddition, a vacuum system 356 may be provided The vacuum system 356 mayinclude a vacuum inducing portion or member 358, such as a resilientbulb. The vacuum inducing member 358 may be interconnected with the tube12 through a selected connecting portion 360.

The vacuum connecting portion 360 may interconnect with an orifice 362.The orifice 362 may be interconnected or extend from the cap 18 orprovided in any appropriate portion with the tube 12. Nevertheless, afirst one-way valve 364 may be provided along the connection portion 360or near the orifice 362. The first one-way valve 364 provides that aflow of a fluid, such as a gas, may pass in a first direction but not ina second. A second one-way valve 366 may also be provided downstreamfrom the first one-way valve 364. In this way, a vacuum may be createdwith the vacuum inducing member 358, such that air is drawn out of thetube 12 and removed through the second one-way valve 366 in thedirection of arrow V. Due to the first and second one-way valves 364,366 the air is generally withdrawn from the tube 12 withoutsubstantially allowing the air to flow back into the tube 12. Thus, avacuum can be created within the tube 12 to assist with removing aselected volume of fluid, such as blood, from the patient 350.

Because the tube 12 may be filled substantially directly from thepatient 350, the collection of the fluid, such as blood, may be providedsubstantially efficiently to the tube 12. Although any appropriatemechanism may be used to assist in withdrawing the blood from thepatient 350 the vacuum system 356 may be provided including the vacuuminducing member 358. Any appropriate vacuum creating device may be used,such as a mechanical pump or the like. Nevertheless, the tube 12 may befilled for use during a selected procedure.

As discussed above, the tube 12 may be used to separate a selectedportion of the blood obtained from the patient 350 substantiallyintraoperatively. Therefore, the collection or separation of the variouscomponents may be substantially autologous and substantiallyintraoperatively. Moreover, obtaining the fluid directly from thepatient 350 may increase the efficiency of the procedure and theefficiency of the intraoperative or the operative procedure.

With reference to FIG. 9, the separator 10 may be used to separate anyappropriate material. The material may be separated for any purpose,such as a surgical procedure. For example, a selected fraction of a bonemarrow aspirate or a bone marrow portion may be produced with theseparator 10 according to various embodiments. The selected fraction ofthe bone marrow aspirate may include various components, such asundifferentiated cells. The various undifferentiated cells may bepositioned in a selected scaffold or relative to a selected portion of apatient for providing a volume of the undifferentiated cells to thepatient. As known to those skilled in the art, a selected portion of thepatient may include bone, cartilage, connective tissue, or any othertissue. Also as known by those skilled in the art, a selected portion ofthe patient may include a defect in bone, cartilage, connective tissueand/or any other tissue. It will be understood that the method describedaccording to FIG. 9 is merely exemplary of various embodiments that maybe used to provide a selected fraction of a bone marrow aspirate orother material to a patient or selected position. The selected portionmay be placed on the scaffold in any appropriate manner, such as byspraying, dipping, infiltrating, or any appropriate method.

A method of selecting or creating a selected fraction of a bone marrowaspirate in a selected scaffold according to a method 400 is illustratedin FIG. 9. Generally, the method 400 may start in block 402 in obtaininga bone marrow aspirate volume. The bone marrow aspirate (BMA) may beobtained in any selected or generally known manner. For example, aselected region of bone, such as a portion near an operative procedure,may be used to obtain the bone marrow aspirate. Generally, an accessingdevice, such as a syringe and needle, may be used to access anintramedullary area of a selected bone. The BMA may then be withdrawninto the syringe for various procedures. Once a selected volume of theBMA is obtained in block 402, the BMA may be positioned in the separator10 according to various embodiments in block 404. The BMA may bepositioned in any appropriate separator, such as those described aboveincluding the separator 10. Once the BMA is positioned in the separator10, a selected fraction of the BMA may be separated from the BMA inblock 406.

BMA is a complex tissue comprised of cellular components (thatcontribute to bone growth) including red and white blood cells, theirprecursors and a connective tissue network termed the stroma. Bonemarrow stromal cells or mesenchymal stem cells have the potential todifferentiate into a variety of identifiable cell types includingosteoblasts, fibroblasts, endothelial cells, reticulocytes, adipocytes,myoblasts and marrow stroma. The selected fraction of the BMA mayinclude undifferentiated cells or any appropriate portion of the BMA.The fractionation or separation of various fractions of the BMA mayallow for a volume of BMA to be taken from a single location and theseparation or concentration of the selected portion may be performed inthe separator 10. Generally, obtaining a small volume of the selectedportion from a plurality of locations may be used to obtain anappropriate volume of BMA or selected fraction of the BMA. Nevertheless,the separator 10 may allow for separating a selected volume from asingle location from which the BMA is obtained. This may reduce the timeof a procedure and increase the efficiency of obtaining the selectedfraction of the BMA.

In addition to obtaining a volume of the BMA in block 402, a volume ofwhole blood may be obtained in block 408. The volume of blood obtainedin block 408, according to any appropriate procedure, including thosedescribed above, may then be positioned in the separator 10, in block410. The whole blood may be positioned in any appropriate separator,such as those described above or a separator to separate a selectedfraction of the whole blood. As described above, the whole blood may beseparated into an appropriate fraction, such as a fraction including aplatelet portion or buffy coat. The whole blood may be separated intoselected fractions in block 412. It will be understood that the BMA andthe whole blood volume may be obtained substantially simultaneously orconsecutively in block 402 and 408. In various embodiments, concentratedBMA, such as the middle fraction 332, 226 comprising nucleated cells inseparator 10 has a concentration of nucleated cells that is at least 4times the concentrate of nucleated cells in BMA. Similarly, the selectedfractions of the BMA obtained in block 406 and whole blood obtained inblock 412 may also be performed substantially sequentially orsimultaneously. For example, the separator 10 including the volume ofthe BMA may be positioned in a separating device, such as a centrifuge,substantially opposite, so as to balance, the separator 10 including thevolume of the whole blood. Therefore, a single separation, such ascentrifuge procedure may be used to separate both the BMA and the wholeblood into selected fractions. This again may increase the efficiency ofthe procedure to provide both a selected fraction of the BMA and aselected fraction of the whole blood substantially simultaneously.

The selected fractions of the BMA and the whole blood, provided in block406 and 412 may be harvested in block 414. The selected fractions of theBMA and the whole blood, may be harvested in block 414 for appropriatepurposes, such as those described herein. The separator 10 may be usedto obtain the selected fractions of the BMA and the whole blood, throughvarious procedures, such as those described above.

A plurality of separators 10 may be used to obtain a larger quantity ofthe selection fractions and such quality of selected fractions may bepooled together. In various embodiments, the BMA aspirate can beconcentrated alone or in combination with whole blood. In variousembodiments, whole blood may be added to the separator 10, and theresulting buffy coat fraction (middle fraction 332, 226) may not onlycontain the at least 4 times greater concentration of nucleated cellsfrom bone marrow, and may include at least 5 times greater concentrationof white blood cells from the whole blood and at least 8 times greaterconcentration in platelets from the whole blood. In addition,circulating stem cells from whole blood may be concentrated with themature white blood cells in the buffy coat.

After harvesting the selected fractions of the BMA and the whole bloodin block 414, the selected fraction of the BMA may be positioned on anappropriate scaffold in block 416. The scaffold in block 416 may be anyappropriate scaffold, such as synthetic bone substitutes or allergenictissue. Examples of a scaffold include, but are not limited to, bone,cartilage, bone substrates, ceramics, biopolymers, collagens, metal, andthe like. The scaffolds may be used for appropriate procedures, such ashard or soft tissue grafting, including uses in non-union or chronicwounds. The undifferentiated cells of the BMA may allow for asubstantial source of cells for use during a substantially naturalhealing after an operative procedure, for example, the natural healingof a patient may use the supplied undifferentiated cells. In such anatural healing of a patient, the undifferentiated cells may be appliedto a wound, a defect, a graft site, a bone, cartilage, connectivetissue. and the like. Therefore, the scaffold may be positioned in aselected portion of the anatomy and the cells may be allowed to grow anddifferentiate into selected portions in the implanted position.

It is well known to those skilled in the art that bone marrow containshematopoietic and mesenchymal stems cells that are precursors for mostof the cells found within the body. In general, the hematopoietic cells,along with angiogeneic growth factors, such as those found in platelets,will promote angiogenesis. Angiogenesis is a necessary stage in woundhealing of most tissues and treatment of ischemia. The mesenchymal stemcells are the cells responsible for the formation of bone, tendon,ligament, articular cartilage, muscle, fat, intervertebral discs,meniscus, skin, and any other structural tissue found in the body. Invarious embodiments, a concentration and delivery of these precursorcells, with or without platelet concentrate, will improve therapeuticuses over the native bone marrow.

In addition to positioning the selected fractioning of the BMA and thescaffold in block 416, the platelets of the whole blood may bepositioned on or near the scaffold of block 418. The platelets of thewhole blood fraction positioned in the scaffold of block 418 may assistthe undifferentiated cells and the anatomy into which the scaffold ispositioned to allow for a substantially efficient and complete healing.The platelet rich fraction of the whole blood sample may include varioushealing and growth factors that may assist in providing an efficient andproper healing in the anatomy. Therefore, the undifferentiated cells ofthe BMA, or other selected fraction obtained from the separation of theBMA, and the selected fraction of the whole blood, obtained from theseparator, may be used with the scaffold to provide a substantiallyefficient implant.

In some embodiments, harvest block 414 may be combined with a growthfactors which may include any of the well-known growth factors such asPlatelet-Derived Growth Factor (PDGF), Transforming Growth Factor Beta(TGF-β), Insulin-Like Growth Factor (IGF), Fibroblast Growth Factor(FGF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor(VEGF), Bone Morphogenetic Proteins (BMPs), and vectors for genetherapy. In various embodiments, harvest block 414 may be combined withcellular solutions, suspensions, and materials including osteoblasts,osteoprogenitor cells, chondroblasts, stem cells, or fibroblasts mayalso be used, as may solutions or suspensions containing othertherapeutic agents such as antibiotics, analgesics, pharmaceuticalagents, antithrombinolytics, or chemotherapeutic agents. In variousembodiments, the buffy coat, platelet fraction, and/or undifferentiatedcell fraction (the middle fraction 332, 226) may be combined with anactivator such as thrombin solutions and the like. In variousembodiments the middle fraction 332, 226 may be used alone for cartilagerepair. In various embodiments, the platelet rich fraction may be usedto fill a cartilage defect with a fibrin matrix, or it may be mixed withother cell sources such as autologous chondrocytes, synovial cells, bonemarrow cells, or to mix with the blood clot formed during microfracture.In addition, the separator 10, or any appropriate separator, such asthat described above, may allow for a substantially quick and efficientseparation of the BMA and the whole blood into an appropriate fractionfor use in the procedure. Other examples include pooling whole bloodand/or BMA from different sites of the anatomy or from differentsources.

In various embodiments, the concentrated bone marrow cells can also beincluded with a carrier to aide in delivery and to help maintain thecells' location after implantation. Examples of carriers can includefibrin, concentrated fibrin, demineralized bone matrix, gelatin,collagen, porous calcium based ceramics, porous metal, synthetic fibermatrices, or resorbable matrices. In addition, carriers can be made fromother autogeneic, allogenic, and xenogeneic tissues.

After the selected portion of the BMA and the whole blood are positionedon the scaffold in blocks 416 and 418 the scaffold may be implanted inblock 420. As described above, the scaffold may be implanted in anyappropriate position in the block 420 for various procedures. It will beunderstood that the scaffold may be implanted for any appropriateprocedure and may allow for positioning the selected portion of the BMA,such as undifferentiated cells, and the selected portion of the wholeblood, such as platelets, relative to a selected portion of the anatomy.The scaffold may allow for a bone ingrowth, such as allowed with theundifferentiated cells, to assist in healing of a selected portion ofthe anatomy. In various embodiments, concentrated bone marrow aspiratecan be used in articular cartilage repair. The middle fraction 332, 226can be added to a focal defect or to an osteoarthritic defect. Thedefects can be in any joint that contains articular cartilage. Invarious embodiments, the middle fraction 332, 226 can promote theformation of repair tissue. In addition, these cells can be added to amicrofracture technique in order to increase the mesenchymal cellspresent in the defect and increase the amount and quality of repairtissue that forms. In various embodiments, the middle fraction 332, 226can be delivered in one of the carriers listed above. In variousembodiments, for delivery to cartilage, the middle fraction 332, 226 inan autologous fibrin or concentrated fibrin is activated with anactivating solution so that it forms a 3-D gel in situ and holds themiddle fraction 332, 226 within the cartilage defect. In variousembodiments, the concentrated bone marrow can also be used in meniscusrepair. The concentrated bone marrow can be used to fill a tear with themeniscus, or in can be used to soak a graft used to replace the meniscusafter a full or partial meniscectomy. In various embodiments,concentrated bone marrow aspirate used in combination with platelet richplasma can also be used for repair of meniscus. In various embodiments,concentrated bone marrow aspirate can be used to repair bone. In variousembodiments, the middle fraction 332, 226 can used alone, or mixed withan appropriate carrier such as demineralized bone matrix, calcium basedceramics, fibrin, or concentrated fibrin. The defects in bone could befound in long bones, cranium, sternum, and spine. One specific placementof concentrated bone marrow aspirate would be to deliver theundifferentiated cells to a freeze dried demineralized bone product(such as the Bonus DBM product from Biomet Biologics) under vacuum. Invarious embodiments, the concentrated bone marrow can infiltrate thegraft, and the plasma in the bone marrow may hydrate the bone matrix andcreate an injectable carrier. The undifferentiated cells may havedifferentiating growth factors included within the demineralized bone tostimulate cartilage and bone formation. In various embodiments, theconcentrated bone marrow can be delivered to the patient with a growthfactor that will induce proliferation, chemotaxis, and/or morphogeneis.Examples of growth factors that could be used include PDGF, TGF-b, IGF,VEGF, EGF, CTGF, FGF, and any of the BMPs.

The description of the invention is merely exemplary in nature and,thus, variations that do not depart from the gist of the invention areintended to be within the scope of the invention. Such variations arenot to be regarded as a departure from the spirit and scope of theinvention.

1. A method for concentrating bone marrow aspirate, the methodcomprising: obtaining a volume of bone marrow aspirate from a mammal;loading the volume of bone marrow aspirate into a separator comprisingtwo buoys, the separator operable to separate the aspirate into three ormore fractions; centrifuging the separator creating at least onefraction comprising a concentrated bone marrow aspirate; and extractingthe at least one fraction comprising a concentrated bone marrowaspirate.
 2. The method according to claim 1 wherein the at least onefraction comprises at least one of the group consisting essentially ofhematopoietic stem cells, stromal stem cells, mesenchymal stem cells,endothelial progenitor cells, red blood cells, white blood cells,fibroblasts, reticulacytes, adipose cells, and endothelial cells.
 3. Themethod according to claim 1 wherein an amount of nucleated cells in theat least one fraction is greater than or equal to 4 times an amount ofnucleated cells found in the volume of bone marrow aspirate.
 4. Themethod according to claim 1 further comprising applying the at least onefraction into a patient.
 5. A method for concentrating bone marrowaspirate and peripheral blood, the method comprising: collecting avolume of bone marrow aspirate and a volume of peripheral blood from apatient; loading the volume of bone marrow aspirate and the volume ofperipheral blood into a separator comprising two buoys, separator beingoperable to separate a combination of the volume of aspirate and thevolume of blood into three or more fractions; centrifuging the at leastone separator operably creating at least one fraction comprising aconcentration of at least one of bone marrow aspirate and peripheralblood; and withdrawing the at least one fraction comprising theconcentration.
 6. The method according to claim 5 wherein theconcentration comprises at least one of the group consisting essentiallyof hematopoietic stem cells, stromal stem cells, mesenchymal stem cells,endothelial progenitor cells, red blood cells, white blood cells,fibroblasts, reticulacytes, adipose cells, and endothelial cells.
 7. Themethod according to claim 5 wherein an amount of nucleated cells in theconcentration is greater than or equal to 4 times an amount of nucleatedcells in the combination of the volume of aspirate and the volume ofblood.
 8. A method for treating a defect in a mammal using aconcentrated bone marrow aspirate, the method comprising: drawing avolume of bone marrow aspirate from the mammal; loading the volume ofbone marrow aspirate into a separator comprising two buoys, theseparator being operable to separate the aspirate into three or morefractions; centrifuging the separator; separating the volume of bonemarrow aspirate into a plurality of fractions including a fractioncomprising a concentrated bone marrow aspirate; extracting the fractioncomprising a concentrated bone marrow aspirate; and applying thefraction comprising a concentrated bone marrow aspirate to site of thedefect.
 9. The method according to claim 8 wherein the fractioncomprising a concentrated bone marrow aspirate is essentially buffy coatincluding a plurality of undifferentiated cells.
 10. The methodaccording to claim 8 wherein the fraction comprises at least one of thegroup consisting of hematopoietic stem cells, stromal stem cells,mesenchymal stem cells, endothelial progenitor cells, red blood cells,white blood cells, fibroblasts, reticulacytes, adipose cells, andendothelial cells.
 11. The method according to claim 8 furthercomprising combining the fraction with a carrier.
 12. The methodaccording to claim 11 wherein the carrier is selected from the groupconsisting essentially of collagen, hydrogel, a bioabsorbable polymer, abiopolymer, water, buffered solution, fibrin, concentrated fibrin,demineralized bone matrix, gelatin, porous calcium based ceramics,porous metal, synthetic fiber matrices, resorbable matrices, autogeneictissue, allogenic tissue, xenogeneic tissue, and combinations thereof.13. The method according to claim 8 further comprising combining anactivating agent with the fraction.
 14. The method according to claim 8further comprising combining a pharmaceutical agent with the fraction.15. A method for treating a defect in an animal, the method comprising:obtaining a volume of bone marrow aspirate and a volume of whole bloodfrom the animal; loading the volume of bone marrow aspirate into a firstseparator comprising two buoys, the separator being operable to separatethe aspirate into three or more fractions; loading the volume of wholeblood into a second separator comprising two buoys, the separator beingoperable to separate the aspirate into three or more fractions;centrifuging the first separator and the second separators, therebyseparating the volume of bone marrow aspirate into a plurality offractions and separating the volume of whole blood into a plurality offractions. collecting a fraction of the bone marrow aspirate; collectinga fraction of the whole blood; and applying at least one of the fractionof bone marrow aspirate and the fraction of whole blood to site of thedefect.
 16. The method according to claim 15 wherein the at least one ofthe fraction of bone marrow aspirate and the fraction of whole bloodcomprises at least one of the group consisting of hematopoietic stemcells, stromal stem cells, mesenchymal stem cells, endothelialprogenitor cells, red blood cells, white blood cells, fibroblasts,reticulacytes, adipose cells, and endothelial cells.
 17. The methodaccording to claim 15 further comprising combining the at least one ofthe fraction of bone marrow aspirate and the fraction of whole bloodwith a carrier.
 18. The method according to claim 17 wherein the carrieris selected from the group consisting essentially of collagen, hydrogel,a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin,concentrated fibrin, demineralized bone matrix, gelatin, porous calciumbased ceramics, porous metal, synthetic fiber matrices, resorbablematrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, andcombinations thereof.
 19. The method according to claim 17 furthercomprising adding an activating agent to the at least one of thefraction of bone marrow aspirate and the fraction of whole blood
 20. Themethod according to claim 17 further comprising adding a pharmaceuticalagent to the at least one of the fraction of bone marrow aspirate andthe fraction of whole blood.
 21. A method for treating a defect in apatient, the method comprising: drawing a volume of bone marrow aspirateand a volume of whole blood from the patient; adding a firstanticoagulant to the volume of bone marrow aspirate; adding a secondanticoagulant to the volume of whole blood; loading the volume of bonemarrow aspirate and the volume of whole blood into a separatorcomprising two buoys, the separator being operable to separate thevolume of bone marrow aspirate and the volume of whole blood into threeor more fractions; centrifuging the separator, separating the volume ofbone marrow aspirate and the volume of whole blood into a plurality offractions. withdrawing a fraction comprising at least one of the groupconsisting of hematopoietic stem cells, stromal stem cells, mesenchymalstem cells, endothelial progenitor cells, red blood cells, white bloodcells, fibroblasts, reticulacytes, adipose cells, and endothelialcells.; and applying the fraction to site of the defect.
 22. The methodaccording to claim 21 wherein the fraction is essentially buffy coatincluding a plurality of undifferentiated cells.
 23. The methodaccording to claim 21 further comprising combining the fraction with acarrier.
 24. The method according to claim 32 wherein the carrier isselected from the group consisting essentially of collagen, hydrogel, abioabsorbable polymer, a biopolymer, water, buffered solution, fibrin,concentrated fibrin, demineralized bone matrix, gelatin, porous calciumbased ceramics, porous metal, synthetic fiber matrices, resorbablematrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, andcombinations thereof.
 25. The method according to claim 21 furthercomprising combining an activating agent with the fraction.
 26. Themethod according to claim 21 further comprising combining apharmaceutical agent with the fraction.
 27. A method of treating apatient with combination of a concentrated bone marrow aspirate andbuffy coat, the method comprising: obtaining a volume of a whole bloodfrom the patient; obtaining a volume of a bone marrow aspirate from thepatient; forming a buffy coat fraction of the whole blood; forming aconcentrated bone marrow aspirate fraction of the bone marrow aspirate;and applying at least one of the buffy coat fraction or the concentratedbone marrow aspirate fraction to the patient.
 28. The method accordingto claim 27, wherein the forming a first fraction of the first wholematerial and the forming a second fraction of the second whole materialincludes: loading the volume of whole blood and the volume of bonemarrow aspirate in a container; and applying a force to the container toform at least three fractions.
 29. The method according to claim 27,wherein the forming a first fraction of the first whole material and theforming a second fraction of the second whole material includes: loadingthe volume of whole blood and the volume of bone marrow aspirate in thecontainer having a separating member, the separating member having aspecific gravity substantially dependent upon at least one of the atleast three fractions; and centrifuging the container to move theseparating member to a selected position relative to the at least one ofthe volume of whole blood and the volume of bone marrow aspirate tosubstantially physically separate the at least three fractions.
 30. Themethod according to claim 27 wherein the at least one of the firstfraction or the second fraction comprises at least one of the groupconsisting of hematopoietic stem cells, stromal stem cells, mesenchymalstem cells, endothelial progenitor cells, red blood cells, white bloodcells, fibroblasts, reticulacytes, adipose cells, and endothelial cells.31. The method according to claim 27 further comprising combining the atleast one of the first fraction or the second fraction with a carrier.32. The method according to claim 31 wherein the carrier is selectedfrom the group consisting essentially of collagen, hydrogel, abioabsorbable polymer, a biopolymer, water, buffered solution, fibrin,concentrated fibrin, demineralized bone matrix, gelatin, porous calciumbased ceramics, porous metal, synthetic fiber matrices, resorbablematrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, andcombinations thereof.